Method of promoting regulatory T-cell proliferation

ABSTRACT

Described herein are immunosuppressive molecules including immunosuppressive variants of IL-2, and use of such molecules to treat inflammatory and autoimmune disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/672,417, filed Nov. 8, 2012, which is a continuation of U.S. patentapplication Ser. No. 13/145,537, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No. PCT/US2010/021519(which designated the United States), having an international filingdate of Jan. 20, 2010, which is incorporated herein by reference in itsentirety, which claims priority to U.S. Provisional Patent ApplicationNo. 61/146,111, filed Jan. 21, 2009.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1474-US-CNT_Seqlisting_ST25.txt, created Nov. 7, 2012, which is 13 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND

IL-2 binds three transmembrane receptor subunits: IL-2Rβ and IL-2Rγwhich together activate intracellular signaling events upon IL-2binding, and CD25 (IL-2Rα) which serves to present IL-2 to the other 2receptor subunits. The signals delivered by IL-2Rβγ include those of thePI3-kinase, Ras-MAP-kinase, and STAT5 pathways.

T cells require expression of CD25 to respond to the low concentrationsof IL-2 that typically exist in tissues. T cells that express CD25include both CD4⁺ FOXP3⁺ regulatory T cells (T-reg cells)—which areessential for suppressing autoimmune inflammation—and FOXP3⁻ T cellsthat have been activated to express CD25. FOXP3⁻ CD25⁺ T effector cells(T-eff) may be either CD4⁺ or CD8⁺ cells, both of which can bepro-inflammatory and may contribute to autoimmunity, organ graftrejection or graft-versus-host disease. IL-2-stimulated STAT5 signalingis crucial for normal T-reg cell growth and survival and for high FOXP3expression.

Because of the low affinity IL-2 possesses for each of the three IL-2Rchains, a further reduction in affinity for IL-2Rβ and/or IL-2Rγ couldbe offset by an increased affinity for CD25. Mutational variants of IL-2have been generated that exhibit up to 170-fold higher affinity for CD25(US Patent Application Publication No. 2005/0142106; Rao et al.,Biochemistry 44, 10696-701 (2005)). These variants were reported toassociate for several days with cell surface CD25 and to chronicallypromote growth of an IL-2-dependent cell line. The authors report thatthe mutants stimulate persistent T cell growth and, thus, may be usefulin methods of viral immunotherapy and in treating cancer or otherhyperproliferative disorders. High doses of IL-2 (Proleukin) areadministered to cancer patients to induce anti-tumor immunity, atreatment that is often associated with undesirable toxicity. U.S. Pat.No. 6,955,807 describes IL-2 variants that are said to have reducedtoxicity. The patent attributes the toxicity to IL-2-induced stimulationof natural killer (NK) cells, which only express IL-2Rβ and IL-2Rγ. TheIL-2 variants described therein were said to have reduced toxicitybecause they selectively activate CD25⁺ T cells over NK cells. Again theIL-2 variants were said to be useful in therapeutic methods wherein itis beneficial to generally stimulate the immune system, e.g., thetreatment of cancer or infectious diseases.

SUMMARY

Provided herein are immunosuppressive mutational variants of IL-2 thatpreferentially promote the growth/survival of FOXP3⁺ regulatory T cells(T-reg cells) over the growth/survival of potentially proinflammatoryFOXP3⁻ CD25⁺ T cells. By increasing the ratio of T-reg to other T cellsand/or by increasing FOXP3 expression in T-reg without activating FOXP3⁻CD25⁺ T cells, these variants should suppress undesirable inflammation.

The unique properties of these IL-2 variants stem from two sets ofmutations. One set of mutations results in a reduced affinity for thesignaling chains of the IL-2 receptor (IL-2Rβ/CD122 and/orIL-2Rγ/(CD132) and/or a reduced capacity to induce a signaling eventfrom one or both subunits of the IL-2 receptor. The second set ofmutations confers higher affinity for CD25 (IL-2Rα) and may includemutations described by Rao et al. (US Patent Application Publication No.2005/0142106).

As described herein, certain IL-2 variants induce signaling events thatpreferentially induce survival, proliferation, activation and/orfunction of T-reg cells. In certain embodiments, the IL-2 variantretains the capacity to stimulate, in T-reg cells, STAT5 phosphorylationand/or phosphorylation of one or more of signaling molecules downstreamof the IL-2R, e.g., p38, ERK, SYK and LCK. In other embodiments, theIL-2 variant retains the capacity to stimulate, in T-reg cells,transcription or protein expression of genes or proteins, such as FOXP3or IL-10, that are important for T-reg cell survival, proliferation,activation and/or function. In other embodiments, the IL-2 variantexhibits a reduced capacity to stimulate endocytosis of IL-2/IL-2Rcomplexes on the surface of CD25⁺ T cells. In other embodiments, theIL-2 variant demonstrates inefficient, reduced, or absence ofstimulation of PI3-kinase signaling, such as inefficient, reduced orabsent phosphorylation of AKT and/or mTOR (mammalian target ofrapamycin). In yet other embodiments, the IL-2 variant retains theability of wt IL-2 to stimulate STAT5 phosphorylation and/orphosphorylation of one or more of signaling molecules downstream of theIL-2R in T-reg cells, yet demonstrates inefficient, reduced, or absentphosphorylation of STAT5, AKT and/or mTOR or other signaling moleculesdownstream of the IL-2R in FOXP3⁻ CD4⁺ or CD8⁺ T cells or NK cells. Inother embodiments, the IL-2 variant is inefficient or incapable ofstimulating survival, growth, activation and/or function of FOXP3⁻ CD4⁺or CD8⁺ T cells or NK cells.

Provided are methods of treating an inflammatory or autoimmune disorder.The methods comprise administering a therapeutically effective amount ofone or more immunosuppressive IL-2 variants to a subject.

Further provided are methods of promoting proliferation, survival,growth, or activation of regulatory T cells. The methods comprisingcontacting a FOXP3-positive (POXP3⁺) T cell with an immunosuppressiveIL-2 variant.

Also provided is the use of an immunosuppressive IL-2 variant in thepreparation of a medicament for the treatment of an inflammatory orautoimmune disorder.

Also provided are compositions of IL-2 variants conjugated to additionalprotein sequences or other chemical that prolong the stability and/orhalf-life of the therapeutic in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Sequences of IL-2 variants. Sequences that vary from germlinehuman IL-2 are not shaded in gray.

FIGS. 2A-2F. FIG. 2A An example of flow cytometric data and gatingstrategy. Representative data is shown in FIG. 1A where 9.5% of CD4⁺cells are CD25⁺FOXP³⁺, 9.9% of CD4⁺ cells are CD25⁺FOXP3⁻ and 6.7% ofCD8⁺ cells are CD25⁺FOXP³⁻. FOXP3⁺CD8⁺ T cells are typically veryinfrequent. FIG. 2B. Relative number of CD8⁺CD25⁺FOXP3⁻ T cells. FIG.2C. Relative number of CD4⁺CD25⁺FOXP3⁻ T cells. FIG. 2D. Relative numberof CD4⁺CD25⁺FOXP3⁺ T cells. FIG. 2E. Ratio of FOXP3⁺/FOXP3⁻ cells amongCD25⁺CD4⁺ T cells. FIG. 2F. IL-2-mediated FOXP3 upregulation inFOXP3⁺CD4⁺ T cells.

FIG. 3 . IL-2 muteins stimulate phospho-STAT5 in T-reg but areinefficient at stimulating signals in other T cells. T cells werestimulated with IL-2 as described in Example 3. Phospho-STAT5 wasmeasured by flow cytometry and phospho-AKT was measured by ELISA(MesoScale Discovery). Abbreviations are as follows: T-reg, FOXP3⁺CD4⁺ Tcells; CD4 T-eff, CD4⁺CD25⁺FOXP3⁻ “effector” T cells; CD8 T-eff,CD8⁺CD25⁺FOXP3⁻ “effector” T cells.

DESCRIPTION OF PREFERRED EMBODIMENTS

FOXP3⁺ regulatory T cells (T-reg cells) are essential for maintainingnormal immune homeostasis and immune tolerance to self tissues, as wellas for suppressing undesirable inflammation. T-reg cells exert theirsuppressor and regulatory functions through multiple mechanisms whichare likely to be regulated by temporal and environmental factors.Current immunosuppressive therapeutics generally target individualproinflammatory pathways and as such often exhibit partial efficacy orare applicable to specific diseases. An alternative immunosuppressivemodality might involve the elevation of the numbers and activation stateof natural suppressor cells to better enable them to deliver appropriatesuppressor molecules/activities at sites of inflammation.

Described herein are therapeutic agents that selectively promote T-regcell proliferation, survival, activation and/or function. By“selectively promote,” it is meant the therapeutic agent promotes theactivity in T-reg cells but has limited or lacks the ability to promotethe activity in non-regulatory T cells. Further described herein areassays to screen for agents that selectively promote T-reg cellproliferation, survival, activation and/or function. Agents that may bescreened include, but are not limited to, small molecules, peptides,polypeptides, proteins including antibodies, e.g., monoclonal,humanized, human, monovalent, bivalent, and multivalent antibodies.

In certain embodiments, the agent is an IL-2 variant. In particular, theIL-2 variant promotes these activities of T-reg cell growth/survival buthave a reduced ability, as compared to wild-type IL-2, to promotenon-regulatory T-cell (FOXP3⁻ CD25⁺) and NK cell proliferation,survival, activation and/or function. In certain embodiments, such IL-2variants function through a combination of elevated affinity for theIL-2R subunit IL-2Rα (CD25) and a reduced affinity for the signalingsubunits IL-2Rβ and IL-2Rγ. Whereas IL-2 and variants thereof have beenused in the art as immunostimulatory agents, e.g., in methods oftreating cancer or infectious diseases, the IL-2 variants describedherein are particularly useful as immunosuppressive agents, e.g., inmethods of treating inflammatory disorders.

IL-2 variants comprise a sequence of amino acids at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93% at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% identical to wild-type IL-2. IL-2variants further include a sequence of amino acids at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93% at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identical to a functional fragmentof wild-type IL-2. As used herein, “wild-type IL-2” shall mean thepolypeptide having the following amino acid sequence:

(SEQ ID NO: 1) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT wherein X is C, S, A or V.

Variants may contain one or more substitutions, deletions, or insertionswithin the wild-type IL-2 amino acid sequence. Residues are designatedherein by the one letter amino acid code followed by the IL-2 amino acidposition, e.g., K35 is the lysine residue at position 35 of SEQ ID NO:1.Substitutions are designated herein by the one letter amino acid codefollowed by the IL-2 amino acid position followed by the substitutingone letter amino acid code, e.g., K35A is a substitution of the lysineresidue at position 35 of SEQ ID NO:1 with an alanine residue.

In one aspect, the invention provides immunosuppressive IL-2 variantsthat have a higher affinity for IL-2Rα than wild-type IL-2. U.S.Published Patent Application No. 2005/0142106 (incorporated herein byreference in its entirety) describes IL-2 variants that have higheraffinity for IL-2Rα than does wild-type IL-2 and methods of making andscreening for such variants. Preferred IL-2 variants contain one or moremutations in positions of the IL-2 sequence that either contact IL-2Rαor alter the orientation of other positions contacting IL-2Rα, resultingin higher affinity for IL-2Rα. The mutations may be in or near areasknown to be in close proximity to IL-2Rα based on published crystalstructures (Xinquan Wang, Mathias Rickert, K. Christopher Garcia.Science 310:1159 2005). IL-2 residues believed to contact IL-2Rα includeK35, R38, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, L72, andY107.

IL-2 variants having greater affinity for IL-2Rα can include a change inN29, N30, Y31, K35, T37, K48, E68, V69, N71, Q74, S75, or K76. Preferredvariants include those having one or more of the following mutations:N29S, N30S, N30D, Y31H, Y31S, K35R, T37A, K48E, V69A, N71R, and Q74P.

Immunosuppressive IL-2 variants also include variants that demonstratealtered signaling through certain pathways activated by wild-type IL-2via the IL-2R and result in preferentialproliferation/survival/activation of T-reg. Molecules known to bephosphorylated upon activation of the IL-2R include STAT5, p38, ERK,SYK, LCK, AKT and mTOR. Compared to wild-type IL-2, theimmunosuppressive IL-2 variant can possess a reduced PI3K signalingability in FOXP3⁻ T cells, which can be measured by a reduction in thephosphorylation of AKT and/or mTOR as compared to wild-type IL-2. Suchvariants may include mutations in positions that either contact IL-2Rβor IL-2Rγ or alter the orientation of other positions contacting IL-2Rβor IL-2Rγ. IL-2 residues believed to contact IL-2Rβ include L12, Q13,H16, L19, D20, M23, R81, D84, S87, N88, V91, I92, and E95. IL-2 residuesbelieved to contact IL-2Rγ include Q11, L18, Q22, E110, N119, T123,Q126, S127, I129, S130, and T133. In certain embodiments, the IL-2variant comprises a mutation at E15, H16, Q22, D84, N88, or E95.Examples of such mutations include E15Q, H16N, Q22E, D84N, N88D, andE95Q.

In certain embodiments, the IL-2 variant comprises a combination ofmutations. Examples of IL-2 variants having a combination of mutationsare provided in FIG. 1 and include haWT (SEQ ID NO:2), haD, (SEQ IDNO:3), haD.1 (SEQ ID NO:4), haD.2 (SEQ ID NO:5), haD.4 (SEQ ID NO:6),haD.5 (SEQ ID NO:7), haD.6 (SEQ ID NO:8), haD.8 (SEQ ID NO:9), andhaD.11 (SEQ ID NO:10)d. In preferred embodiments, the IL-2 variantstimulates STAT5 phosphorylation in FOXP3-positive regulatory T cellsbut has reduced ability to induce STAT5 and AKT phosphorylation inFOXP3-negative T cells as compared to wild-type IL-2. Preferred variantshaving such properties include haD, haD.1, haD.2, haD.4, haD.5, haD.6,and haD.8.

The IL-2 variants may further comprise one or more mutations as comparedto the wild-type IL-2 sequence that do not have an effect on theaffinity for IL-2Rβ or IL-2Rγ, provided the IL-2 variant promotes thepreferential proliferation, survival, activation or function of FOXP3⁺T-reg over that of other T cells that do not express FOXP3. In preferredembodiments, such mutations are conservative mutations.

The IL-2 variant may comprise one or more compounds to increase theserum-half-life of the IL-2 variant when administered to a patient. Suchhalf-life extending molecules include water soluble polymers (e.g.,polyethylene glycol (PEG)), low- and high-density lipoproteins, antibodyFc (monomer or dimer), transthyretin (TTR), and TGF-β latency associatedpeptide (LAP). Also contemplated are IL-2 variants comprising acombination of serum half-life extending molecules, such as PEGylatedTTR (US Pat. Appl. Publ. No. 2003/0195154).

Methods of Making an Immunosuppressive IL-2 Variant

The immunosuppressive IL-2 variants can be produced using any suitablemethod known in the art, including those described in U.S. Pat. No.6,955,807 for producing immunostimulatory IL-2 variants (incorporatedherein by reference). Such methods include constructing a DNA sequenceencoding the IL-2 variant and expressing those sequences in a suitablytransformed host. This method will produce the recombinant variant ofthis invention. However, the variants may also be produced by chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology. Batch-wise production or perfusion production methods areknown in the art. See Freshey, R. I. (ed), “Animal Cell Culture: APractical Approach,” 2nd ed., 1992, IRL Press. Oxford, England; Mather,J. P. “Laboratory Scaleup of Cell Cultures (0.5-50 liters),” MethodsCell Biolog 57: 219-527 (1998); Hu, W. S., and Aunins, J. G.,“Large-scale Mammalian Cell Culture,” Curr Opin Biotechnol 8: 148-153(1997); Konstantinov, K. B., Tsai, Y., Moles, D., Matanguihan, R.,“Control of long-term perfusion Chinese hamster ovary cell culture byglucose auxostat.,” Biotechnol Prog 12:100-109 (1996).

In one embodiment of a recombinant method for producing a variant, a DNAsequence is constructed by isolating or synthesizing a DNA sequenceencoding the wild type IL-2 and then changing one or more codons bysite-specific mutagenesis. This technique is well known. See, e.g., Market. al., “Site-specific Mutagenesis Of The Human Fibroblast InterferonGene”, Proc. Natl. Acad. Sci. USA 81, pp. 5662-66 (1984); and U.S. Pat.No. 4,588,585, incorporated herein by reference.

Another method of constructing a DNA sequence encoding the IL-2 variantwould be chemical synthesis. This for example includes direct synthesisof a peptide by chemical means of the protein sequence encoding for anIL-2 variant exhibiting the properties described herein. This method mayincorporate both natural and unnatural amino acids at positions thataffect the interactions of IL-2 with the IL2Rα, IL-2Rβ, or IL-2Rγ.Alternatively, a gene which encodes the desired IL-2 variant may besynthesized by chemical means using an oligonucleotide synthesizer. Sucholigonucleotides are designed based on the amino acid sequence of thedesired IL-2 variant, and preferably selecting those codons that arefavored in the host cell in which the recombinant variant will beproduced. In this regard, it is well recognized that the genetic code isdegenerate—that an amino acid may be coded for by more than one codon.For example, Phe (F) is coded for by two codons, TTC or TTT, Tyr (Y) iscoded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W)is coded for by a single codon, TGG. Accordingly, it will be appreciatedthat for a given DNA sequence encoding a particular IL-2 variant, therewill be many DNA degenerate sequences that will code for that IL-2variant.

The DNA sequence encoding the IL-2 variant, whether prepared by sitedirected mutagenesis, chemical synthesis or other methods, may or maynot also include DNA sequences that encode a signal sequence. Suchsignal sequence, if present, should be one recognized by the cell chosenfor expression of the IL-2 variant. It may be prokaryotic, eukaryotic ora combination of the two. It may also be the signal sequence of nativeIL-2. The inclusion of a signal sequence depends on whether it isdesired to secrete the IL-2 variant from the recombinant cells in whichit is made. If the chosen cells are prokaryotic, it generally ispreferred that the DNA sequence not encode a signal sequence. If thechosen cells are eukaryotic, it generally is preferred that a signalsequence be encoded and most preferably that the wild-type IL-2 signalsequence be used.

Standard methods may be applied to synthesize a gene encoding an IL-2variant. For example, the complete amino acid sequence may be used toconstruct a back-translated gene. A DNA oligomer containing a nucleotidesequence coding for an IL-2 variant may be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide may be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the DNA sequences encoding an IL-2 variant will be insertedinto an expression vector and operatively linked to an expressioncontrol sequence appropriate for expression of the IL-2 variant in thedesired transformed host. Proper assembly may be confirmed by nucleotidesequencing, restriction mapping, and expression of a biologically activepolypeptide in a suitable host. As is well known in the art, in order toobtain high expression levels of a transfected gene in a host, the genemust be operatively linked to transcriptional and translationalexpression control sequences that are functional in the chosenexpression host. The choice of expression control sequence andexpression vector will depend upon the choice of host. A wide variety ofexpression host/vector combinations may be employed.

Any suitable host may be used to produce the IL-2 variant, includingbacteria, fungi (including yeasts), plant, insect, mammal, or otherappropriate animal cells or cell lines, as well as transgenic animals orplants. More particularly, these hosts may include well known eukaryoticand prokaryotic hosts, such as strains of E. coli, Pseudomonas,Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodopterafrugiperda (Sf9), animal cells such as Chinese hamster ovary (CHO) andmouse cells such as NS/O, African green monkey cells such as COS 1, COS7, BSC 1, BSC 40, and BNT 10, and human cells, as well as plant cells intissue culture. For animal cell expression, CHO cells and COS 7 cells incultures and particularly the CHO cell line CHO (DHFR−) or the HKB lineare preferred.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences described herein. Neither will all hosts function equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevectors copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, preferredvectors for use in this invention include those that allow the DNAencoding the IL-2 variants to be amplified in copy number. Suchamplifiable vectors are well known in the art. They include, forexample, vectors able to be amplified by DHFR amplification (see, e.g.,Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, “Construction Of AModular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals UtilizedFor Efficient Expression”, Mol. Cell. Biol., 2, pp. 1304-19 (1982)) orglutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No.5,122,464 and European published application 338,841).

The IL-2 variants may be glycosylated or unglycosylated depending on thehost organism used to produce the variant. If bacteria are chosen as thehost, then the IL-2 variant produced will be unglycosylated. Eukaryoticcells, on the other hand, will glycosylate the IL-2 variant, althoughperhaps not in the same way as native IL-2 is glycosylated. The IL-2variant produced by the transformed host can be purified according toany suitable method. Various methods are known for purifying IL-2. See,e.g., Current Protocols in Protein Science, Vol. 2. Eds: John E.Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T.Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc).

The biological activity of the IL-2 variants can be assayed by anysuitable method known in the art. Such assays include those described inthe Examples below.

Indications

Diseases, disorders, or conditions may be amenable to treatment with ormay be prevented by administration of a T-reg-selective IL-2 variant toa subject. Such diseases, disorders, and conditions include, but are notlimited to, inflammation, autoimmune disease, paraneoplastic autoimmunediseases, cartilage inflammation, fibrotic disease and/or bonedegradation, arthritis, rheumatoid arthritis, juvenile arthritis,juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoidarthritis, polyarticular juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), juvenile dermatomyositis, juvenile psoriatic arthritis,juvenile scleroderma, juvenile systemic lupus erythematosus, juvenilevasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemiclupus erythematosus, vasculitis, myolitis, polymyolitis,dermatomyolitis, osteoarthritis, polyarteritis nodosa, Wegener'sgranulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis,scleroderma, sclerosis, primary biliary sclerosis, sclerosingcholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttatepsoriasis, inverse psoriasis, pustular psoriasis, erythrodermicpsoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus,Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis,inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis,celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barredisease, Type I diabetes mellitus, thyroiditis (e.g., Graves' disease),Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD,transplantation rejection, and the like. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of a T-reg-selective IL-2 variant are provided.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. A T-reg-selective IL-2 variant need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient A T-reg-selective IL-2 variant in an amountand for a time sufficient to induce a sustained improvement overbaseline of an indicator that reflects the severity of the particulardisorder.

Pharmaceutical Compositions

In some embodiments, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of one or a plurality ofT-reg-selective IL-2 variants of the invention together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In addition, the invention providesmethods of treating a patient by administering such pharmaceuticalcomposition. The term “patient” includes human and animal subjects.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolality, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, sucrose,mannose or dextrins); proteins (such as serum albumin, gelatin orimmunoglobulins); coloring, flavoring and diluting agents; emulsifyingagents; hydrophilic polymers (such as polyvinylpyrrolidone); lowmolecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, PEG, sorbitan esters,polysorbates such as polysorbate 20, polysorbate, triton, tromethamine,lecithin, cholesterol, tyloxapol); stability enhancing agents (such assucrose or sorbitol); tonicity enhancing agents (such as alkali metalhalides, preferably sodium or potassium chloride, mannitol sorbitol);delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

The therapeutically effective amount of T-reg-selective IL-2variant-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the T-reg-selective IL-2 variant is being used,the route of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient.

In certain embodiments, the clinician may titer the dosage and modifythe route of administration to obtain the optimal therapeutic effect. Atypical dosage may range from about 0.1 μg/kg to up to about 30 mg/kg ormore, depending on the factors mentioned above. In specific embodiments,the dosage may range from 0.1 μg/kg up to about 30 mg/kg, optionallyfrom 1 μg/kg up to about 30 mg/kg or from 10 μg/kg up to about 5 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular T-reg-selective IL-2 variant in the formulation used.Typically, a clinician administers the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, or as two or more doses (which may ormay not contain the same amount of the desired molecule) over time, oras a continuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

Combination Therapies

In further embodiments, T-reg-selective IL-2 variant is administered incombination with other agents useful for treating the condition withwhich the patient is afflicted. Examples of such agents include bothproteinaceous and non-proteinaceous drugs. When multiple therapeuticsare co-administered, dosages may be adjusted accordingly, as isrecognized in the pertinent art. “Co-administration” and combinationtherapy are not limited to simultaneous administration, but also includetreatment regimens in which a T-reg-selective IL-2 variant isadministered at least once during a course of treatment that involvesadministering at least one other therapeutic agent to the patient.

In certain embodiments, a T-reg-selective IL-2 variant is administeredin combination with an inhibitor of the PI3-K/AKT/mTOR pathway, e.g.,rapamycin (rapamune, sirolimus). Inhibitors of this pathway incombination with IL-2 favor T-reg enrichment.

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

EXAMPLES Example 1 Panel of IL-2 Mutants

To examine the potential for generating IL-2 variants with reducedcapacity to stimulate FOXP3⁻ CD25⁺ “effector” T cells (T-eff) but notT-reg, a series of IL-2 mutants was generated in which amino acidspredicted to interact with the IL-2Rβ and/or IL-2Rγ chain were altered.These variants also contained a set of previously described mutationsthat conferred high affinity for CD25 (variant “2-4” in Rao et al.,Biochemistry 44, 10696-701 (2005)). This series of variants is shown inFIG. 1 . Variant haWT contained only the mutations that contributed tothe high affinity for CD25. Variants haD, haD.1, haD.2, etc, alsocontained mutations predicted to alter interactions with IL-2Rβ and/orIL-2Rγ. In all assays, variant haD.11 was not capable of inducing anysignal or altering any cellular phenotype and, as such, served as acontrol for CD25 binding without IL-2R signaling. All the IL-2 variantscontained the C125S mutation for improved manufacturability andterminated with FLAG and HIS-tag sequences (DYKDDDDKGSSHHHHHH) (SEQ IDNO:11)

Several assays were used to assess the ability of the IL-2 variants toinduce signaling events and T cell growth. These included assays todetect:

1. Growth and survival of T cell subsets and measurement of FOXP3expression.

2. Cell signaling (e.g. detection of phosphorylated STAT5 and AKT usingflow cytometric and ELISA-based methods).

Example 2 Enrichment of FOXP3⁺ Cells and Retention of FOXP3 UpregulationDuring Long Term T Cell Culture

Total PBMC were activated in 24-well plates at 4×10⁶ cells per well with100 ng/ml anti-CD3 (OKT3). On day 3 of culture, cells were washed 3times and rested in fresh media for 3 days. Cells were then washed andseeded in 96 well flat-bottom plates with IL-2 variants at either 10 nMor 100 pM. Three days later cells were counted and analyzed by flowcytometry. (FIG. 2A)

As expected, CD8⁺CD25⁺ T cells were especially responsive to WT IL-2 andvariant haWT, however, all variants that contained mutated IL-2Rβ and/orγ contact residues were very inefficient at promoting accumulation ofactivated CD8⁺CD25⁺ T cells (FIG. 2B). A similar trend was observed forCD4⁺CD25⁺FOXP3⁻ T cells (FIG. 2C). In contrast, the growth/survival ofFOXP3⁺ CD4⁺ T cells was stimulated by several IL-2 variants to a degreesimilar to that of WT IL-2 (FIG. 2D). As a result, the ratio of FOXP3⁺to FOXP3⁻ T cells among CD4⁺ CD25⁺ T cells was increased by several IL-2variants with IL-2βγ-contact residue mutations (FIG. 2E). Furthermore,the mutations did not impair IL-2-stimulated FOXP3 upregulation in T-reg(FIG. 2F).

Example 3 Mutations that Reduce Signaling in FOXP3⁻ T Cells butStimulate STAT5 Signaling in T-Reg

The IL-2 variants were screened for their ability to stimulate AKT andSTAT5 phosphorylation in T cell subsets. Several IL-2 variants were aspotent, or nearly as potent, as wt IL-2 at stimulating STAT5 in FOXP3⁺ Tcells 10 min after stimulation. Three hours after washing IL-2 from themedia, some IL-2 variants (haD, haD.1, haD.2, haD.4, haD.6, and haD.8)continued to stimulate sustained STAT5 signaling at levels higher thanthat seen with wt IL-2. In contrast, for FOXP3⁻ T cells, STAT5 and AKTresponses to the haD variants after 10 min stimulation were not nearlyas high as those stimulated by wt IL-2 or haWT. After 3 hrs, weak STAT5and AKT signals similar to those seen with wt IL-2 were observed inT-eff, however, at this late timepoint wt IL-2 signaling had diminishedgreatly. In FOXP3⁺ T cells, AKT signaling is not normally stimulated byIL-2 (Zeiser R, et al, 2008 Blood 111:453) thus the phospho-AKT signalobserved in total T cell lysates can be attributed to T-eff.

Methods: Previously activated (with anti-CD3 for 2-3 days) and rested(in fresh culture medium for 2-5 days) T cells were exposed to 1 nM wtor mutant IL-2 for 10 min at 37° C. Cells were then stained (10 mintimepoint) or washed and cultured for an additional 3 hrs (3 hrtimepoint). To measure phospho-AKT by ELISA, a 50 μl culture was stoppedby adding an equal volume of 2× lysis buffer and lysates were measuredfor phospho-AKT with multiplex ELISA plates according to themanufacturer's protocols (MesoScale Discovery, Gaithersburg, Md.). Tomeasure phospho-STAT5 by flow cytometry, a 50 μl culture was stopped byadding 1 ml of FOXP3 Fix/Perm Buffer (BioLegend, San Diego, Calif.),incubation at 25° C. for 20 min, and staining for cell surface markers,FOXP3 and phospho-STAT5 according the BioLegend FOXP3 staining protocol.

The invention claimed is:
 1. A method of promoting regulatory T-cellproliferation in a subject, said method comprising administering to asubject in need thereof a therapeutically effective amount of an IL-2variant having a mutation in a residue that contacts IL-2Rβ, whereinsaid IL-2 variant comprises a N88D substitution, a substitution in aresidue that contacts IL-2Rα selected from the group consisting of N29S,Y31H, K35R, T37A, K48E, V69A, N71R, and Q74P, and a sequence of aminoacids at least 95% identical to SEQ ID NO:1, wherein the IL-2 varianthas a reduced ability, as compared to wild-type IL-2, to promotenon-regulatory T-cell growth, and wherein the IL-2 variant induces STAT5phosphorylation in ex vivo FOXP3-positive T cells comprising afunctional IL-2 receptor complex but has a reduced ability to inducephosphorylation of STAT5 in FOXP3-negative T cells, wherein saidadministration of a therapeutically effective amount of the IL-2 variantresults in preferential proliferation of regulatory T cells.
 2. Themethod of claim 1, wherein the IL-2 variant promotes FOXP3-positiveregulatory T cell growth or survival in vitro.
 3. The method of claim 1,wherein the IL-2 variant is conjugated to a chemical or polypeptide thatextends the serum half-life of said IL-2 variant in vivo.
 4. The methodof claim 1, wherein the mutation that contacts IL-2Rα is V69A.
 5. Themethod of claim 1, wherein the subject has asthma, diabetes, allergy,systemic lupus erythematosus, ankylosing spondylitis, vasculitis,Sjogren's syndrome, inflammatory bowel disease, Crohn's disease,ulcerative colitis, celiac disease, multiple sclerosis, COPD, Type Idiabetes mellitus, organ graft rejection or graft-versus-host disease.